Project Summary/Abstract Hematopoietic stem cell (HSC) transplantations are used to treat a wide variety of hematological and non- hematological malignancies. However, a major barrier to widespread clinical use is limiting donor stem cell numbers for transplantation. To combat this issue, HSCs expansion for therapeutic use has been a major clinical effort for decades. However most attempts have met with minimal success, as enforcing proliferation ex vivo has led to functional decline of these cells upon transplantation into a host. Here, my proposal focuses on understanding how proliferative signals in vivo impact HSC regenerative potential through changes in gene expression analysis. In the lab, we take advantage of a tet-off double transgenic mouse model that allows us to progressively track accumulation of HSC cell divisions over time. Using this model, we demonstrated that during homeostasis progressive accumulation of cell division events resulted in the progressive loss of regenerative potential. Furthermore, we showed that infrequently dividing label-retaining HSCs contained all, if not most, of the long- term regenerative potential within the bone marrow. Continuing on these findings, we set out to enforce HSC proliferation in vivo using three different proliferative stimuli: progressive aging, myeloablative stress, and inflammation. Preliminary data demonstrates that enforced proliferation through progressive aging, or by treatment with the chemotherapeutic 5-flurouracil (5-FU), demonstrated differential effects on HSC regenerative potential: progressive proliferation through aging causes progressive regenerative decline, while proliferation enforced by 5-FU does not alter HSC regenerative potential. To better understand how proliferation alters HSC regenerative potential, we adopted a two-pronged approach. First we will enforce HSCs to proliferate with three clinically relevant stimuli: aging, myeloablation, and inflammation. Then we will assay functional changes due to proliferation through serial transplantation. Second, we will conduct gene expression analysis of changes that occur due to proliferation through RNAseq. This will allow us to couple functional regenerative output due to enforced proliferation with gene expression changes for each proliferative stimulus. By studying the functional and genetic consequences of proliferation, we hope to understand how to induce the appropriate self-renewal signals that will promote the expansion of HSCs in vitro without the loss of regenerative potential.